Phosphoinositides function as cytoplasmic signaling lipids that are recognized by protein domains. In this proposal we characterize the structure and binding sites of the FYVE domain, a signaling domain that specifically recognizes phosphatidylinositol 3-phosphate (PtdIns(3)P). The interaction of FYVE domains and PtdIns(3)P is critical for a range of biological processes including membrane trafficking, signal transduction, and cytoskeletal organization. This project focuses on the structure and function of the FYVE domain of early endosome autoantigen (EEA1), the prototypic and best characterized member of the FYVE family of proteins. The fusion of early endosome membranes is mediated by the EEA1 protein, allowing ligand:receptor complexes to be internalized and sorted for degradation or recycling back to the cell surface. Our hypotheses are: l: The FYVE domain has a unique 3D structure: The FYVE domain is defined by a unique and highly conserved sequence of 75 residues of unknown structure. The solution structure of the FYVE domain of human EEA1 is being solved by heteronuclear magnetic resonance (NMR) spectroscopy to identify structural and functional features conserved within the FYVE domain family that distinguish it from known folds. 2: A PtdIns(3)P binding site is conserved among FYVE domains: The PtdIns(3)P binding site and kinetic properties of wild type and mutant FYVE domain will be characterized by NMR chemical shifts changes, nuclear Overhauser effects, in vitro liposome binding assays, and in vivo cellular localization assays. The specificity of the interaction will be established by comparing interactions with different phosphoinositides. 3: The FYVE domain's oligomeric state influences its function: EEA1 is known to form parallel dimers, impacting its interactions with PtdIns(3)P-containing membranes and Rab5 protein complexes. We show that the EEA1 FYVE domain dimerizes, and are defining the structure and functional properties of monomers and dimers using pulsed field gradient and half-filtered NMR, sedimentation equilibrium, and activity assays. 4: A unique zinc coordination site stabilizes the PtdIns(3)P site: FYVE domains coordinate two zinc ions through eight conserved cysteines. The specificity and structure of the zinc site is characterized by NMR using chelators, various divalent cations, and cysteine mutations. Zinc's structural importance is demonstrated by FYVE domain refolding experiments, and is related to other zinc binding domains such as RING fingers.